EP0837930B1 - A TRP-226 MUTANT HUMAN GLANDULAR KALLIKREIN-1 (hK2) - Google Patents
A TRP-226 MUTANT HUMAN GLANDULAR KALLIKREIN-1 (hK2) Download PDFInfo
- Publication number
- EP0837930B1 EP0837930B1 EP96920863A EP96920863A EP0837930B1 EP 0837930 B1 EP0837930 B1 EP 0837930B1 EP 96920863 A EP96920863 A EP 96920863A EP 96920863 A EP96920863 A EP 96920863A EP 0837930 B1 EP0837930 B1 EP 0837930B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- trp
- protein
- arg
- prepro
- expression vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to novel proteins.
- it relates to a novel form of human glandular kallikrein-1 (hK2) encoded by a newly-identified hK2 gene and use of recombinant DNA techniques to obtain proteins encoded by the hK2 genes, including the mature protein encoded by the previously recognised hK2 gene, Arg 226 -hK2, as an active protein.
- hK2 human glandular kallikrein-1
- the human glandular kallikrein gene family is composed of genes encoding three different proteins: prostate specific antigen (PSA), glandular kallikrein-1 (hK2) and pancreatic/renal kallikrein (KLK1). These genes are located on chromosome 19 and the PSA and hK2 genes are aligned at a distance of 12 kb(1).
- the similarity of the coding region of the human KLK1 gene to the coding regions of the human PSA and K2 genes is 74% and 75% respectively.
- the coding regions of the hPSA and hK2 genes are 85% homologous and the promoter regions of the same genes are 91% homologous.
- the KLK1 gene encodes the true kallikrein.
- KLK1 has a kininogenase activity and it is expressed in kidney, pancreas and salivary gland (2).
- the hPSA and hK2 genes have been shown to be expressed only in prostatic epithelial cells. Despite the similarity of the hK2 and hPSA genes, the expression level of the hK2 gene at mRNA level is only about 10-30% of that of the hPSA gene in the prostate (3).
- LNCaP cells clear up-regulation of the mRNA levels for both hK2 and hPSA was observed in the presence of androgens (4).
- hK2 and hPSA are not in fact prostate specific as previously believed.
- Southern blot analysis with gene-specific oligonucleotide probes after RT-PCR expression of all three previously identified human kallikrein genes has been detected in the human endometrium (5).
- hPSA has also been detected in milk of lactating women by immunoassay (6). It has additionally been found that 30-40% of breast tumours as well as steroid hormone stimulated normal breast tissue samples contain hPSA (7).
- hK2 gene has been isolated from a human genomic fetal liver DNA library and sequenced (8a). The nucleotide sequence of the five coding exons of this hK2 gene was found to encode a 261 amino acid preproprotein with a signal peptide of 17 amino acids and an activation peptide of 7 amino acids like PSA. hK2 has been found to possess the typical catalytic triad (His41-Asp96-Ser189) of serine proteases. The presence of an aspartate residue at amino acid position 183 in hK2 fits with a trypsin-like substrate activity for this protein. In PSA, there is a serine residue at the same position which accounts for PSA in contrast exhibiting chymotrypsin-like activity (8).
- PSA The function of PSA is the cleavage of semenogelin clots (9), but the function of hK2 is unknown.
- concentration of PSA in serum is increased in cancer and hyperplasia of the prostate gland and PSA is widely used as a marker for the detection and monitoring of prostate cancer (10).
- an hK2 protein has, however, remained to be purified. Also, the purity of PSA is not always unambiguous. This leads to problems with hPSA assays that are based on polyclonal or monoclonal antibodies raised against hPSA.
- an hK2 protein has now been obtained free of hPSA contamination by cloning hK2 cDNA from a human prostate cancer tissue cDNA library in an expression vector and expressing the cDNA in appropriate host cells. More specifically, an hK2 protein has been produced by providing a baculovirus expression vector encoding a prepro-hK2 protein in insect cells (see Examples 4 and 5).
- baculovirus expression vector system for production of an hK2 protein
- a recombinant Autographa California Nuclear Polyhedrosis virus AcNPV
- a prepro-hK2 protein for example, to transform bacterial cells, e.g. E. coli cells
- the pre-pro-sequence will not be removed by post-translational processing to give directly the corresponding mature protein, e.g. active mature Arg 226 -hK2.
- an hK2 cDNA By cloning an hK2 cDNA from a human prostate cDNA library, an hK2 cDNA has now been identified which has one base difference from the coding sequence for hK2 previously reported. This difference at base position 792 (C to T) equates with an amino acid change at amino acid position 226 from Arg to Trp. That this observed base difference was not an artifact of the cDNA cloning and sequencing procedure but reflects the existence of a previously unidentified hK2 gene was confirmed by using PCR to amplify the hK2 gene from genomic DNA of a number of human prostate and leucocyte samples (see Example 2).
- the present invention thus provides a protein which is Trp 226 -hK2 having a sequence identical to Arg 226 -hK2 apart from change of Arg 226 to Trp or which is Trp 226 -hK2 having an N- and/or C-terminal extension and which retains the ability to bind Trp 226 -hK2 antibodies or hPSA antibodies, with the proviso that where said protein is a naturally-occurring protein it is substantially free of other proteins with which it is ordinarily associated.
- Such proteins include, in addition to Trp 226 -hK2 in substantially pure form, for example, proTrp 226 -hK2 or prepro-Trp 226 -hK2 in substantially pure form wherein Trp 226 -hK2 is joined at the N-terminus to the pro- or prepro-sequences previously identified for Arg 226 -hK2.
- a further novel protein forming an embodiment of the present invention is Trp 226 -hK2 having the N-terminal dipeptide extension Ser-Arg.
- Trp 226 -hK2 rather than Trp 226 -hK2 per se, is obtained by expression of a cDNA encoding prepro-Trp 226 -hK2 in insect cells. It is detectable using a conventional hPSA immunoassay as commercially available for use in clinical studies thus demonstrating cross-reactivity with known anti-hPSA antibodies, but is inactive (see Example 3).
- the present invention additionally extends to fragments of Trp 226 -hK2 proteins as hereinbefore described which retain antigenicity and the Trp 226 amino acid residue. All such fragments and any protein having the Trp 226 -hK2 sequence are included within the term Trp 226 -hK2 protein used hereinafter.
- the present invention provides an isolated DNA or recombinant DNA encoding a Trp 226 -hK2 protein of the invention.
- a DNA may include the natural coding sequence for Trp 226 -hK2, more preferably the complete natural coding sequence for pre-pro-Trp 226 -hK2.
- Such a recombinant DNA may be in the form of a vector, e.g. a plasmid or viral vector.
- a vector may be an expression vector which when present in host cells is capable of directing production of a Trp 226 -hK2 protein of the invention in said cells or the culture medium.
- a baculovirus expression vector which when present in insect cells is capable of directing production of a Trp 226 -hK2 protein of the invention in the culture medium or in said cells.
- the invention provides host cells containing a vector of the invention as hereinbefore described and a method of preparing a Trp 226 -hK2 protein of the invention which comprises culturing host cells of the invention containing an expression vector under conditions whereby said protein is produced in the host cells or the culture medium and isolating said protein from said cells or medium.
- said cells are insect cells containing a baculovirus expression vector for expression in the cells of prepro-Trp 226 -hK2
- Trp 226 -hK2 with the N-terminal dipeptide extension Ser-Arg may be purified from the culture medium.
- the purification protocol for this purpose may, for example, comprise a combination of ion-exchange chromatography and gel filtration chromatography steps (see Example 5).
- the Trp 226 -hK2 derivative thus obtained may readily be subsequently converted to Trp 226 -hK2 using techniques well known to those skilled in the art of protein engineering.
- active Arg 226 -hK2 may alternatively be isolated from the culture medium of host insect cells e.g. by a purification protocol comprising a combination of ion-exchange chromatography and gel filtration chromatography steps (see Example 5).
- active Arg 226 -hK2 substantially free of other proteins with which it is ordinarily associated and more particularly active Arg 226 -hK2 in substantially pure form.
- active Arg 226 -hK2 is meant a protein having the mature Arg 226 -hK2 sequence and capable of hydrolyzing the synthetic chromogenic polypeptide substrate H-D-Pro-Phe-Arg-pNA.2HCl, where pNA is paranitroaniline, in an assay procedure as described in Example 5. Active Arg 226 -hK2 initially thus obtained may be subsequently digested to antigenic fragments. Isolation of Arg 226 -hK2 or an antigenic fragment thereof may be followed by labelling of the protein.
- a protein of the present invention may be labelled with any label conventionally employed for labelling proteins.
- a protein of the present invention may be labelled with a radiolabel, an enzyme label (e.g. alkaline phosphatase), a fluorescent label (e.g. fluorescein or rhodamine) a lanthanide or biotin (which may be detected by avidin or streptavidin conjugated to peroxidase).
- a protein of the present invention may find use as a standard reference or to test for antibody reactivity to the protein in an appropriate immunoassay or immunohistochemical assay. Such tests may include, for example, testing for cross-reactivity to an antibody raised against hPSA, Arg 226 -hK2 or Trp 226 -hK2.
- a protein of the present invention in unlabelled form may find use as an immunogen for the production of polyclonal or monoclonal antibodies.
- the protocol employed for polyclonal or monoclonal antibody production may conform with any of the conventional procedures known for antibody production.
- the protein will preferably be combined with an adjuvant, e.g. Freund's complete adjuvant, in an immunising composition.
- a suitable procedure for monoclonal antibody production to Trp 226 -hK2 or Arg 226 -hK2 may conform, for example, with the procedure described in Clinical Chemistry (1987) 33, 103-107 for obtaining monoclonal antibodies to human prostatic acid phosphatase or the procedure described in Clinical Chemistry (1990) 26, 92-95 for obtaining monoclonal antibodies to human prostate-specific antigen.
- An antibody so obtained may also be labelled in conventional manner, e.g. with a radioactive label, an enzyme label, a fluorescent label, a lanthanide or biotin.
- Trp 226 -hK2 as an immunogen for monoclonal antibody production and subsequently screening hybridomas using purified Trp 226 -hK2 and commercially available purified hPSA
- monoclonal antibodies can be selected which are capable of binding Trp 226 -hK2, but not hPSA in human body samples and which are thus particularly valuable for diagnostic purposes.
- Arg 226 -hK2, or a combination of Arg 226 -hK2 and Trp 226 -hK2 may alternatively be employed in such a procedure as the immunogen.
- Arg 226 -hK2 may be used in addition to, or instead of, Trp 226 -hK2 in the screening protocol.
- Such an antibody screening protocol wherein both Arg 226 -hK2 and Trp 226 -hK2 are employed may be designed to select non-hPSA binding antibodies capable of distinguishing between Arg 226 -hK2 and Trp 226 -hK2.
- the present invention provides an antibody, either in labelled or unlabelled form, capable of binding Trp 226 -hK2 but not hPSA or Arg 226 -hK2.
- the invention additionally provides hybridomas capable of producing such an antibody.
- Trp 226 -hK2 and/or Arg 226 -hK2 may be specifically detected in a sample, e.g. a body sample.
- a sample e.g. a body sample.
- antibody as used herein will be understood to include both complete antibody molecules and antigen-binding fragments thereof such as Fab and F(ab') 2 fragments. Humanised antibodies and fragments thereof are also included within the term "antibody”.
- the Trp 226 -hK2 protein sequence may serve as a marker for prostatic disease, e.g. for diagnosing cancer or hyperplasia of the prostate gland.
- the present invention provides a method of diagnosing prostatic disease in patients homozygous or heterozygous for the Trp 226 -hK2 gene, which comprises determining whether there is altered expression or altered concentration of a protein encoded by said gene in prostate tissue or in human body fluids.
- Fig. 1a to 1d Silver-stained polyacrylamide gel electrophoresis and immunoblot analysis of recombinant hK2 protein.
- the pure recombinant Trp 226 -hK2 protein (lane 1) and the commercial hPSA (lane 2) were silver-stained in native (1a) and reduced SDS-PAGE (1b).
- Rabbit polyclonal antibody raised against hPSA purified from seminal fluid was used to detect recombinant Arg 226 -hK2 (lane 1), recombinant Trp 226 -hK2 (lane 2 and commercial hPSA (lane 3) on blotted native PAGE (1c) and reduced SDS-PAGE (1d).
- Fig. 2A and 2B Quantitative recoveries of recombinant Trp 226 -hK2 and commercial hPSA by a time-resolved fluoroimmunoassay (1A) and IRMA (1B).
- Pure Trp 226 -hK2 and commercial hPSA were diluted with the bovine serum albumin containing zero buffers of the kits to concentrations of 1, 10, 25, 50, 100 and 500 ⁇ l. These concentrations were assayed by the time-resolved fluoroimmunoassay and IRMA kits for hPSA.
- the data are means ⁇ SD from three to five separate determinations.
- Example 1 Cloning of an hK2-cDNA from a human prostate cancer tissue cDNA library
- hK2 cDNA was amplified from a human prostate cancer tissue cDNA library by PCR.
- the N-terminal oligomer was (5'-TCCCCCGGGAGATCTCACCATG-TGGGACCTGGTTCTC-3') and it contained SmaI and BgIII restriction sites.
- the C-terminal oligomer was (5'-CGCTCTA-GATCAGGGGTTGGCTGCGATGGT-3') and contained an Xbal restriction site in addition to the partial hK2 sequence.
- the prepro-hK2 cDNA was inserted into the BgIII/XbaI site of the transfer vector pVL1392 (Invitrogen).
- the coding sequence of this cDNA differed from the human DNA coding sequence for hK2 previously reported by Schedlich et al . (8a) in that coding position 792 was T rather than C.
- Example 2 Sequence analysis of hK2 genes in human prostate tissue and leukocyte samples - detections of the Arg226Trp-polymorphism
- Genomic DNA was isolated from human prostate tissue obtained by prostatectomy, biopsy or transurethral resection, and from human blood leukocytes. Female and young male blood leukocyte DNAs were used as control material. Specific oligonucleotides were used for PCR amplification and sequencing of the hK2 gene. For amplification, the N-terminal oligomer was 5'TTCTCACTGTGTCTCTCCTC-C-3' and the biotin-labelled C-terminal oligomer was 5'G-TGGGACAGGGGCACTCA-3'. For PCR direct sequencing, the fluorescein amidite labelled oligomer was 5'ATCATGGGGCCC-TGAGCC-3'.
- the protein concentration of purified recombinant Trp 226 -hK2 was estimated by the method of Lowry et al. (J. Biol. Chem. 1951; 193:265) with bovine serum albumin (Bio-Rad, Richmond, CA) as the standard.
- the recoveries of recombinant Trp 226 -hK2 were further measured by time-resolved fluoroimmunoassay (DELFIA PSA kit, Wallac, Finland) and IRMA (Tandem®-R PSA kit, Hybritech Europe, Belgium).
- DELFIA PSA kit time-resolved fluoroimmunoassay
- IRMA Tandem®-R PSA kit, Hybritech Europe, Belgium.
- the recombinant Trp 226 -hK2 and commercial PSA were diluted with the "zero" standards of immunoassay kits containing bovine serum albumin.
- the assays intended to measure serum hPSA concentrations were not specific to hPSA, but detected 100 % of inactive hK2 added.
- the active recombinant hK2 was equally detectable in commercial fluoroimmunoassay and IRMA for hPSA.
- Example 4 Cloning of prepro-hK2 cDNA in a baculovirus expression vector and expression in insect cells
- Insect cells containing either a coding sequence for prepro-Trp 226 -hK2 or prepro-Arg 226 -hK2 were found to secrete into the culture medium protein capable of detection by the hPSA assay, although higher expression was observed with the prepro-Trp 226 -hK2 coding sequence.
- the harvested medium from the recombinant virus infection was concentrated with a Pellicon cassette system (cutoff, 10 kDa, Millipore) and dialyzed into 50mM sodium acetate buffer (pH 5.5).
- the concentrate was loaded onto a cation-exchange column (S-Sepharose HP 35/100, Pharmacia). After washing, the hK2 protein was eluted from the column with a linear salt gradient from 0.1 M to 0.25 M NaCl.
- the purity of recombinant Trp 226 -hK2 protein thus obtained was evaluated by SDS-PAGE, native PAGE and isoelectric focusing either by silver-staining or immunostaining (12).
- the molecular weight (Mr) of the recombinant Trp 226 -hK2 protein was found to be approximately 33 kDa (12) while the Mr of commercial hPSA was found to be 34 kDa.
- the detected average Mr was 27.4 kDa.
- silver-stained native PAGE Fig.
- Trp 226 -hK2 protein showed one band of 370 kDa while commercial hPSA showed four bands between 70 and 140 kDa.
- the heterogeneity seen with hPSA was possibly due to endoproteolytic cleavage of the protein into 2 or 4 polypeptide chains held together by disulphide bridges (13).
- N-terminal amino acid analysis showed that posttranslational processing did not completely remove the preprosequence of prepro-Trp 226 -hK2 in the insect cells.
- PSA predominately exists as a complex with ⁇ 1-antichymotrypsin (15).
- Serine protease inhibitors like ⁇ 1-antichymotrypsin and ⁇ 1-antitrypsin usually react with the active site of the proteinase (16). It has been found, however, that recombinant Trp 226 -hK2 protein obtained as above does not complex with either of these serpins.
- the purification procedure given above may also be applied to recover Arg 226 -hK2 from a culture medium.
- insect cells containing a prepro-Arg 226 -hK2 coding sequence in an AcNPV vector operably-linked to a polyhedrin promoter have been shown to produce an active hK2 protein with trypsin-like activity.
- the immunostained native PAGE of the recovered Arg 226 -hK2 protein showed a different pattern when compared to the recombinant Trp 226 -hK2 protein (Fig. 1b and 1c).
- purified recombinant hK2 proteins obtained by the above procedure and purified mature Trp 226 -hK2, e.g. obtained by further processing of a Trp 226 -hK2 derivative of the type discussed above, can be used as antigens for monoclonal antibody production.
- mature Trp 226 -hK2 or mature Arg 226 -hK2 for monoclonal antibody production in accordance with the procedure described by Höyhtyä et al in Clin. Chem.
- a monoclonal antibody capable of distinguishing hK2 from hPSA in samples including human body samples, e.g. samples derived from human prostatic tissue.
- Such an antibody may be desirably labelled with Eu atoms as described in Vihko et al. Clinical Chemistry (1990) 26, 92-95.
Abstract
Description
Claims (26)
- A protein which is Trp226- human glandular kallikrein-1 (Trp226-hK2) having a sequence identical to Arg226-hK2 apart from change of Arg226 to Trp, or which is Trp226-hK2 having an N- and/or C- terminal extension and which retains the ability to bind Trp226-hK2 antibodies or human prostate specific antigen antibodies, with the proviso that where said protein is a naturally-occurring protein it is in pure form and substantially free of other proteins with which it is ordinarily associated.
- A protein according to claim 1 selected from pro-Trp226-hK2 or prepro-Trp226-hK2 wherein Trp226-hK2 is joined at the N-terminus to the pro- or prepro-sequences of prepro-Arg226-hK2.
- The protein according to claim 1 which is Trp226-hK2 having the N-terminal dipeptide extension Ser-Arg.
- An antigenic fragment of a protein according to any one of claims 1 to 3 which retains the Trp226 amino acid residue of Trp226-hK2, with the proviso that where said protein is a naturally-occurring protein it is substantially free of other proteins with which it is ordinarily associated.
- An isolated DNA encoding a protein as claimed in any one of claims 1 to 4.
- A recombinant DNA comprising the DNA as claimed in claim 5.
- A recombinant DNA as claimed in claim 6, which is in the form of a vector.
- A vector as claimed in claim 7 which is an expression vector wherein a coding sequence for a protein as claimed in any one of claims 1 to 4 is operably-linked to a promoter sequence capable of directing expression of said coding sequence in insect cells.
- An expression vector as claimed in claim 8 which is a baculovirus expression vector wherein a coding sequence for a protein as claimed in any one of claims 1 to 4 is operably-linked to a promoter sequence capable of directing expression of said coding sequence in insect cells.
- A baculovirus expression vector as claimed in claim 9, which contains a coding sequence for prepro-Trp226-hK2 under the control of a promoter capable of directing expression of prepro-Trp226-hK2 in insect cells.
- A baculovirus expression vector as claimed in claim 10 which is a recombinant AcNPV vector wherein the coding sequence for prepro-Trp226-hK2 is under the control of the polyhedrin promoter.
- A host cell containing a vector as claimed in any one of claims 7 to 11.
- A method of preparing a protein as claimed in any one of claims 1 to 4, which comprises culturing host cells containing an expression vector as claimed in any one of claims 8 to 11 under conditions whereby said protein is produced in the host cells or the culture medium and isolating said protein from said cells or medium.
- A method as claimed in claim 13 wherein insect cells containing a baculovirus expression vector as claimed in claim 11 or claim 12 are employed and Trp226-hK2 with the N-terminal dipeptide extension Ser-Arg is isolated from the culture medium.
- A method as claimed in claim 14 wherein said isolation comprises ion-exchange chromatography and gel filtration chromatography.
- The method as claimed in claim 14 or 15 for preparing Trp226-hK2, further comprising converting said Trp226-hK2 derivative to Trp226-hK2.
- Use of a protein as claimed in any one of claims 1 to 4 as an immunogen to obtain antibodies capable of binding said protein.
- Use of a protein as claimed in any one of claims 1 to 4 as an immunogen to obtain a monoclonal antibody capable of binding said protein.
- Use of a protein as claimed in claim 17 or claim 18 wherein said antibodies are labelled.
- A protein as claimed in any one of claims 1 to 4 which is labelled.
- Use of a protein as claimed in any one of claims 1 to 4 or 20 as a standard reference or to test for antibody reactivity to said protein in an immunoassay or immunohistochemical assay.
- A method of diagnosing prostatic disease in patients, which comprises determining whether there is altered expression or altered concentration of a protein encoded by the Trp226-hK2 gene in prostate tissue or in the human body fluids.
- A process for preparing an antibody capable of binding Trp226-hK2, but not hPSA or Arg226-hK2, said process comprising using Trp226-hK2 as an immunogen.
- The process as claimed in claim 23, wherein the antibody is labelled.
- Use of an antibody prepared by a process as claimed in claim 23 or claim 24 to detect Trp226-hK2 in a sample.
- A hybridoma capable of producing a monoclonal antibody, which does not bind hPSA and is capable of distinguishing between Arg226-hK2 and Trp226-hK2.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9513281 | 1995-06-29 | ||
GB9513281A GB2302874B (en) | 1995-06-29 | 1995-06-29 | Novel proteins |
PCT/FI1996/000382 WO1997001630A1 (en) | 1995-06-29 | 1996-06-28 | HUMAN GLANDULAR KALLIKREIN-1 (hK2) |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0837930A1 EP0837930A1 (en) | 1998-04-29 |
EP0837930B1 true EP0837930B1 (en) | 2005-01-05 |
Family
ID=10776879
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP96920863A Expired - Lifetime EP0837930B1 (en) | 1995-06-29 | 1996-06-28 | A TRP-226 MUTANT HUMAN GLANDULAR KALLIKREIN-1 (hK2) |
Country Status (12)
Country | Link |
---|---|
US (1) | US6303361B1 (en) |
EP (1) | EP0837930B1 (en) |
JP (1) | JPH11508442A (en) |
AT (1) | ATE286533T1 (en) |
AU (1) | AU712917B2 (en) |
CA (1) | CA2224319A1 (en) |
CZ (1) | CZ417897A3 (en) |
DE (1) | DE69634153D1 (en) |
GB (1) | GB2302874B (en) |
NO (1) | NO976061L (en) |
PL (1) | PL184557B1 (en) |
WO (1) | WO1997001630A1 (en) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6103237A (en) * | 1993-07-22 | 2000-08-15 | Hybritech Incorporated | Stable variant hK2 polypeptide |
EP0974057B1 (en) * | 1996-07-15 | 2005-09-21 | Mayo Foundation For Medical Education And Research | Methods to detect hk2 polypeptides |
US6479263B1 (en) | 1996-11-14 | 2002-11-12 | Baylor College Of Medicine | Method for detection of micrometastatic prostate cancer |
AU8259798A (en) | 1997-06-20 | 1999-01-04 | George G Klee | Method for detection of breast cancer |
FI980488A (en) * | 1998-03-04 | 1999-09-05 | Arctic Partners Oy Ab | New diagnostic method |
CA2360073C (en) | 1999-01-28 | 2011-01-18 | Gen-Probe Incorporated | Probes and primers for detecting prostate specific antigen (psa) in a biological sample |
US20030207808A1 (en) * | 1999-02-18 | 2003-11-06 | Kinneret Savitzky | Novel nucleic acid and amino acid sequences |
EP1159429A2 (en) * | 1999-02-18 | 2001-12-05 | Compugen Ltd. | Psa and klk-2 splicing variants |
US20020151029A1 (en) * | 1999-11-23 | 2002-10-17 | Holloway James L. | Human serine protease |
EP2205249B1 (en) | 2007-09-28 | 2018-11-07 | Intrexon Corporation | Therapeutic gene-switch constructs and bioreactors for the expression of biotherapeutic molecules, and uses thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5516639A (en) * | 1993-07-22 | 1996-05-14 | Mayo Foundation For Medical Education And Research | Antibodies specific for human prostate glandular kallkrein |
WO1995030758A1 (en) | 1994-05-10 | 1995-11-16 | Mayo Foundation For Medical Education And Research | Recombinant hk2 polypeptide |
-
1995
- 1995-06-29 GB GB9513281A patent/GB2302874B/en not_active Expired - Fee Related
-
1996
- 1996-06-28 DE DE69634153T patent/DE69634153D1/en not_active Expired - Lifetime
- 1996-06-28 PL PL96324179A patent/PL184557B1/en not_active IP Right Cessation
- 1996-06-28 CA CA002224319A patent/CA2224319A1/en not_active Abandoned
- 1996-06-28 EP EP96920863A patent/EP0837930B1/en not_active Expired - Lifetime
- 1996-06-28 JP JP9504194A patent/JPH11508442A/en active Pending
- 1996-06-28 AT AT96920863T patent/ATE286533T1/en not_active IP Right Cessation
- 1996-06-28 WO PCT/FI1996/000382 patent/WO1997001630A1/en active IP Right Grant
- 1996-06-28 US US08/983,075 patent/US6303361B1/en not_active Expired - Fee Related
- 1996-06-28 AU AU62273/96A patent/AU712917B2/en not_active Ceased
- 1996-06-28 CZ CZ974178A patent/CZ417897A3/en unknown
-
1997
- 1997-12-23 NO NO976061A patent/NO976061L/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
SAEDI M.S. ET AL: "Overexpression of human prostate specific glandular kallikrein, hK2, in E. coli and generation of antibodies", MOLECULAR AND CELLULAR ENDOCRINOLOGY, vol. 109, 1 April 1995 (1995-04-01), pages 237 - 241 * |
Also Published As
Publication number | Publication date |
---|---|
PL324179A1 (en) | 1998-05-11 |
DE69634153D1 (en) | 2005-02-10 |
CA2224319A1 (en) | 1997-01-16 |
ATE286533T1 (en) | 2005-01-15 |
NO976061D0 (en) | 1997-12-23 |
AU6227396A (en) | 1997-01-30 |
PL184557B1 (en) | 2002-11-29 |
NO976061L (en) | 1998-02-11 |
GB2302874B (en) | 1999-11-10 |
CZ417897A3 (en) | 1998-06-17 |
EP0837930A1 (en) | 1998-04-29 |
JPH11508442A (en) | 1999-07-27 |
GB2302874A (en) | 1997-02-05 |
US6303361B1 (en) | 2001-10-16 |
AU712917B2 (en) | 1999-11-18 |
WO1997001630A1 (en) | 1997-01-16 |
GB9513281D0 (en) | 1995-09-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Lilja et al. | Prostate specific antigen predominantly forms a complex with alpha1‐antichymotrypsin in blood. Implications for procedures to measure prostate specific antigen in serum | |
Christensson et al. | Complex formation between protein C inhibitor and prostate‐specific antigen in vitro and in human semen | |
Magklara et al. | Characterization of the enzymatic activity of human kallikrein 6: autoactivation, substrate specificity, and regulation by inhibitors | |
JP4205738B2 (en) | Stable HK2 polypeptide variant | |
EP0837930B1 (en) | A TRP-226 MUTANT HUMAN GLANDULAR KALLIKREIN-1 (hK2) | |
Becker et al. | Individual prostate-specific antigen (PSA) forms as prostate tumor markers | |
CA2353314C (en) | Mutants of the factor vii-activating protease and detection methods using specific antibodies | |
CA2286090C (en) | Forms of prostate specific antigen and methods for their detection | |
Herrala et al. | Androgen‐sensitive human prostate cancer cells, LNCaP, produce both N‐terminally mature and truncated prostate‐specific antigen isoforms | |
US6284873B1 (en) | Complex of human kallikrein 2 (hK2) and protease inhibitor-6 (PI-6) in prostate tumor tissue and methods of using the complex | |
US6140468A (en) | Recombinant human prostate specific antigen | |
EP0974057B1 (en) | Methods to detect hk2 polypeptides | |
AU737659B2 (en) | Method for detection of metastatic prostate cancer | |
WO1998002748A9 (en) | Methods to detect hk2 polypeptides | |
US20030166036A1 (en) | Protease and an aminopeptidase associated with development of benign prostatic hyperplasia (BPH) | |
CHRISTENSSON et al. | in zyxwvutsrqponmlkji | |
Herrala | Human prostate-specific antigen and glandular kallikrein 2: production and characterization of the recombinant proteins, and association with prostate cancer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 19971224 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB IT LI NL SE |
|
17Q | First examination report despatched |
Effective date: 20020620 |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: ORION DIAGNOSTICA OY |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
RTI1 | Title (correction) |
Free format text: A TRP-226 MUTANT HUMAN GLANDULAR KALLIKREIN-1 (HK2) |
|
RTI1 | Title (correction) |
Free format text: A TRP-226 MUTANT HUMAN GLANDULAR KALLIKREIN-1 (HK2) |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: VIHKO, PIRKKO |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AT BE CH DE DK ES FI FR GB IT LI NL SE |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: NL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20050105 Ref country code: LI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20050105 Ref country code: IT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT;WARNING: LAPSES OF ITALIAN PATENTS WITH EFFECTIVE DATE BEFORE 2007 MAY HAVE OCCURRED AT ANY TIME BEFORE 2007. THE CORRECT EFFECTIVE DATE MAY BE DIFFERENT FROM THE ONE RECORDED. Effective date: 20050105 Ref country code: FR Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20050105 Ref country code: FI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20050105 Ref country code: CH Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20050105 Ref country code: BE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20050105 Ref country code: AT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20050105 |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
REF | Corresponds to: |
Ref document number: 69634153 Country of ref document: DE Date of ref document: 20050210 Kind code of ref document: P |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20050405 Ref country code: DK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20050405 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: DE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20050406 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: ES Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20050416 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: GB Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20050628 |
|
NLV1 | Nl: lapsed or annulled due to failure to fulfill the requirements of art. 29p and 29m of the patents act | ||
REG | Reference to a national code |
Ref country code: CH Ref legal event code: PL |
|
PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
26N | No opposition filed |
Effective date: 20051006 |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
GBPC | Gb: european patent ceased through non-payment of renewal fee |
Effective date: 20050628 |
|
EN | Fr: translation not filed |